Influence of Hurdle Technology On Foodborne Pathog

microorganisms
Article
Influence of Hurdle Technology on Foodborne Pathogen
Survival in the Human Gastrointestinal Tract
Theodora Akritidou, Simen Akkermans, Cindy Smet , Fien de Mey and Jan F. M. Van Impe *
BioTeC+, Chemical and Biochemical Process Technology and Control, Department of Chemical Engineering,
KU Leuven, 9000 Ghent, Belgium
* Correspondence: jan.vanimpe@kuleuven.be
Abstract: The application of several sublethal stresses in hurdle technology can exert microbial
stress resistance, which, in turn, might enable foodborne pathogens to overcome other types of
lethal stresses, such as the gastrointestinal barriers. The present study evaluated the survival of
Salmonella Typhimurium and Listeria monocytogenes during simulated digestion, following exposure
to combinations of water activity (aw ), pH and storage temperature stresses. The results revealed
that both pathogens survived their passage through the simulated gastrointestinal tract (GIT) with
their previous habituation to certain hurdle combinations inducing stress tolerance. More specifically,
the habituation to a low temperature or to a high pH resulted in the increased stress tolerance of
Salmonella, while for Listeria, the cells appeared stress tolerant after exposure to a high temperature
or to a low pH. Nonetheless, both pathogens expressed increased sensitivity after habituation to
growth-limiting hurdle combinations. The survival of stress-tolerant pathogenic cells in the human
GIT poses major public health issues, since it can lead to host infection. Consequently, further research
is required to obtain a deeper understanding of the adaptive stress responses of foodborne bacteria
after exposure to combinations of sublethal hurdles to improve the existing food safety systems.
Keywords: hurdle concept; in vitro digestion; stress adaptation; Salmonella Typhimurium; Listeria
monocytogenes
Citation: Akritidou, T.; Akkermans,
S.; Smet, C.; de Mey, F.; Van Impe,
J.F.M. Influence of Hurdle
Technology on Foodborne Pathogen 1. Introduction
Survival in the Human
The food manufacturing sector has employed a number of practices and resources
Gastrointestinal Tract. Microorganisms
to improve and maintain food safety. Yet, in 2019, more than 300,000 confirmed cases
2023, 11, 405. https://doi.org/
10.3390/microorganisms11020405
of foodborne illnesses have been reported in the EU, with Salmonella enterica and Listeria
monocytogenes occurring among the most frequent agents for human infections [1]. In
Academic Editor: Elena addition, the recent consumer demands for minimally processed, fresh-like and more
González-Fandos natural foods, along with their increased awareness about food safety, put further pressure
Received: 9 January 2023 on the present food safety management systems. A promising response to these concurrent
Revised: 30 January 2023 consumer requests has been the application of the so-called hurdle technology in food
Accepted: 1 February 2023 processing [2,3].
Published: 6 February 2023 The term “hurdle technology” is used to describe the application of more than one
preservative treatment at a low dosage, which are often called hurdles, that extend product
shelf-life without nullifying its nutritional and sensory value [4]. The mode of action of the
hurdle technology relies on combinations of hurdles, with synergistic or additive effects,
Copyright: © 2023 by the authors. aiming at the inhibition or inactivation of targeted microorganisms [2]. In the past, hurdle
Licensee MDPI, Basel, Switzerland. technology was largely used in an empirical manner, considering the lack of profound
This article is an open access article knowledge regarding its governing principles. Nevertheless, nowadays, the intelligent
distributed under the terms and
application of the hurdle concept is highly prevalent in the food industry, since a deeper
conditions of the Creative Commons
understanding of the intrinsic and extrinsic food factors has been established. In the last
Attribution (CC BY) license (https://
decades, the influence of temperature, water activity (aw ), pH, redox potential (Eh) and
creativecommons.org/licenses/by/
competitive microflora, along with their interactions on pathogenic microbial behavior and
4.0/).
Microorganisms 2023, 11, 405. https://doi.org/10.3390/microorganisms11020405 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 405 2 of 19
physiology in food, have been widely studied, amplifying the intelligent use of hurdles in
food manufacturing [5].
Interestingly, despite all the advantages of hurdle technology, there is the potential
for foodborne pathogens to benefit from this food preservation method rather than being
harmed. In view of the sub-inhibitory levels in which each of the combined hurdles is
applied, microbial adaptive stress responses may be triggered that could permit the survival
or even growth of a significant fraction of the microbial population [6]. This phenomenon
describing the ability of a pathogen to respond to harsh environments has often been
characterized as induced tolerance, stress adaptation or “stress hardening” [7]. Bacterial
survival and tolerance in stressful conditions are usually dictated by the microorganism’s
physiological responses, such as modifications occurring in the cell concerning protein
expression and activity, and alterations in the cell membrane and morphology. Furthermore,
these bacterial physiological responses could lead to cross-protection of the pathogen
toward other environmental stresses, inducing increased microbial persistence [6]. An
example of these bacterial adaptive stress responses that is related to microbial protection is
the acid tolerance response (ATR) mechanism, which involves a number of metabolic and
regulatory processes that enable bacteria to survive otherwise lethal stresses. Its activation
depends on the previous exposure of a microbe to a mild stress [8].
The impact of bacterial stress on the stress tolerance and persistence of foodborne
pathogens is a major concern for food safety management, given that stress adaptation
of foodborne pathogens may lead to critical implications for public health, such as the
prolonged survival of pathogens throughout the food chain. In addition, pathogen survival
in the human gastrointestinal tract (GIT) during digestion can be also a catastrophic out-
come of microbial stress adaptation, leading to host infection [9]. The human gut employs
a number of antimicrobial mechanisms to impede pathogen colonization. Upon inges-
tion, a pathogen needs to overcome an assortment of formidable barriers before reaching
an optimal environmental niche to adhere and colonize [10,11]. Gastric acidity is often
characterized as the first line of defense against foodborne pathogens. The gastricidal
action originates from its low pH values, which in the fasted stomach state range from
pH 1.0 to 3.0 [12,13]. In addition, an arsenal of other barriers succeeds the stomach in
the small intestine, including the detergent properties of bile acids, the limited oxygen
availability, the antimicrobial substances secreted by the intestinal epithelium and gut
microbiota. Overcoming all these antimicrobial strategies of the human GIT is the key for
pathogen colonization in the gut and, ultimately, for host infection [11,14].
This paper aims to investigate the impact of the hurdle technology on the survival
patterns of S. Typhimurium and L. monocytogenes in the upper human GIT. To achieve this
goal, a standard meal was contaminated with either of the two pathogens, whose microbial
dynamics were monitored throughout the whole simulated digestion. Different hurdle
combinations of aw , pH and storage temperature were applied for the preparation and
storage of this meal, facilitating the simulation of food processing conditions. Then, after a
storage period of six days under these conditions, the contaminated standard meal was
subdued to an in vitro digestion process, representing the oral, gastric and proximal small
intestinal (duodenum and jejunum) digestion phases.
2. Materials and Methods

2.1. Microorganisms and Preculture Conditions
Salmonella enterica serovar Typhimurium LMG 14933 (isolated from bovine liver)
and Listeria monocytogenes strain LMG23775 (isolated from sausages) were obtained from
the BCCM/LMG bacteria collection of Ghent University in Belgium. The food origin of
the bacterial strains dictated their selection. The stock cultures of S. Typhimurium were
stored frozen (−80 ◦ C) in Tryptone Soy Broth (TSB (Oxoid Ltd., Basingstoke, UK)) with
20% v/v glycerol (Acros Organics, Morris Plains, NJ, USA), whilst the stock cultures of
L. monocytogenes were stored similarly, but in Brain–Heart Infusion (BHI) broth (Oxoid Ltd.,
Basingstoke, UK). With respect to the working cultures, they were renewed every month
and they were stored at 5 ◦ C on Tryptone Soy Agar plates (TSA (Oxoid Ltd., Basingstoke,
UK)) for S. Typhimurium and on BHI agar plates for L. monocytogenes. The preparation of
the precultures was conducted by transferring one colony from the working cultures to a
50 mL Erlenmeyer flask containing 20 mL of TSB for S. Typhimurium and 20 mL of BHI
broth for L. monocytogenes. Then, the flasks were incubated at 37 ◦ C for 24 h until the cells
reached the late stationary phase.
2.2. Inoculum Preparation

For the preparation of the inoculum, an aliquot of the preculture of each microorganism
was used to inoculate the food model system at about 109 CFU (or 20.7 ln(CFU)). For each
experiment, a calculation of the exact volume of this aliquot was implemented, which was
based on a 1:10 dilution of the preculture optical density measurement at 595 nm (reference
absorbances at 595 nm was 0.09 for S. Typhimurium and 0.19 for L. monocytogenes).
2.3. Food Model System Development and Preparation

A food model system (FMS) based on a standard diet was developed, with an energy
(E%) intake of 15% proteins, 35% fat and 50% carbohydrates. The composition of the FMS
was 4.2% w/w whey protein (Power Supplements BV, NL) as a source of proteins, 4.3%
w/w corn oil (Delhaize proxy, Ghent, Belgium) as a source of fat, 13.9% w/w soluble potato
starch (Alfa Aesar, Haverhill, MA, USA) as a source of carbohydrates, NaCl (Sigma-Aldrich,
St. Louis, MO, USA) and water. The total amount of the FMS was 100 g, and the salt
concentration varied to obtain the desired water activity (aw ). In addition, the pH of the
FMS was adjusted according to this research’s demands. All FMSs were constructed as
sterile oil-in-water emulsions [15,16], opting for stable and homogeneous food matrixes. In
addition, all FMSs were supplemented with nonionic surfactants, i.e., Tween 80 (Sigma-
Aldrich, St. Louis, MO, USA) and Span 80 (Sigma-Aldrich, St. Louis, MO, USA). The
storage of each FMS was at 37 ◦ C to preserve the solution’s viscosity, and 100 g of FMS was
transferred aseptically in an empty sterile 1 L Schott bottle the day of the experiment.
2.4. Assessment and Modelling of Boundaries of Growth

As a preliminary task of this study, the growth limits of S. Typhimurium and L. monocy-
togenes were under investigation in order to select the appropriate hurdles to be applied for
the preparation and storage of the FMS. A full factorial experimental design was followed,
where a total of 84 combinations of temperature (10 ◦ C, 25 ◦ C), pH (3.8, 4.2, 4.6, 5.0, 5.6, 5.9,
6.6) and aw (0.909, 0.931, 0.949, 0.957, 0.982, 0.994), in four replicates for each combination,
were examined for both microorganisms by using the FMS as the basal medium. After
autoclaving the FMS, the pH was adjusted aseptically to the desired values using 3 N HCl
(ThermoFisher Scientific, Waltham, MA, USA) or 1 N NaOH (VWR, Radnor, PA, USA).
With respect to aw , NaCl was used to attain the appropriate values, which were measured
with a water activity meter (AWK-40, Nagy, Gäufelden, Germany) after autoclaving the
FMS. The amount of NaCl added in the FMS was decided based on a calibration curve of
aw versus NaCl concentration (conducted in the FMS) that was acquired by a preliminary
experiment. Sterile microcentrifuge vials containing 290 µL of the appropriate FMS for each
combination were inoculated with 10 µL of late stationary phase cells of S. Typhimurium
or L. monocytogenes. The targeted inoculum size was 107 CFU/ mL. Then, the vials were
covered with parafilm to avoid dehydration and stored at 10 ◦ C or 25 ◦ C for 6 days, with
the storage time accounting for food products with a shelf-life of one week. For the deter-
mination of the actual inoculum level of each microorganism, 24 microcentrifuge vials were
sampled immediately after inoculation, and three drops of 20 µL of the appropriate serial
decimal dilution in 0.85% w/v NaCl solution were inoculated on TSA (S. Typhimurium)
or BHI agar (L. monocytogenes) petri dishes. The plates were then incubated at 37 ◦ C for
24 h, and the inoculum level was determined by the enumeration of the colonies. The
assessment of growth in the different combinations of FMS was tested after 6 days of
storage by inoculating three drops of 20 µL of the appropriate serial decimal dilution on
solid general media, as described above. The occurrence of growth was confirmed when
the cell density of the microorganism was higher than the average initial cell density plus
three times its standard deviation.
A mathematical model was used to illustrate the boundary between the experimentally
determined growth and no growth conditions. The four datasets for each combination
of temperature and pathogen were fitted with a separate logistic regression model that
included linear effects and multiplicative interactions between pH and water activity.
2.5. Hurdle Technology Application on the Developed FMS

After the determination of the growth/no growth boundaries of S. Typhimurium and
L. monocytogenes, a two-level factorial design consisting of eight different combinations of
temperature, pH and aw were selected for the preparation and storage of the developed FMS.
These combinations were designated in such a way that four conditions allowed microbial
growth and four conditions inhibited microbial growth (survival) for both microorganisms.
2.6. Enzymes and Bile Acids

The enzymes used during the in vitro digestion process were all purchased from Sigma-
Aldrich (St. Louis, MO, USA) and were α-amylase from hog pancreas (≥50 units/mg
protein) for the oral phase, pepsin from porcine gastric mucosa (≥400 units/mg protein)
for the gastric phase, and pancreatin from porcine and pancreas (8× USP) for the intestinal
phase. Furthermore, bile acids were provided during the intestinal phase with the use of
porcine bile extract (Sigma-Aldrich, St. Louis, MO, USA).
2.7. Simulated Digestion Fluids

Three simulated digestion fluids were used during the in vitro digestion process, one
for each phase of digestion: the Simulated Salivary Fluid (SSF), the Simulated Gastric Fluid
(SGF) and the Simulated Intestinal Fluid (SIF). Their composition was based on the protocol
of INFOGEST [17], and each solution contained the necessary amounts of the following
stock solutions: 0.50 M KCl (VWR, PA, USA), 0.50 M KH2 PO4 (ThermoFisher Scientific, MA,
USA), 1.00 M NaHCO3 (Carl Roth, Karlsruhe, Germany), 2.00 M NaCl, 0.15 M MgCl2 (H2 O)
(Carl Roth, Karlsruhe, Germany), 0.50 M (NH4 )2 CO3 (Carl Roth, Karlsruhe, Germany) and
0.30 M CaCl2 (H2 O)2 (VWR, PA, USA). The latter was added individually in the digesta,
as precipitation occurred when it was added in the simulated digestion stock solutions.
In addition, given that during the digestion process enzymes, calcium chloride and water
were added as well, the simulated digestion fluids were prepared at 1.25× concentration in
order to obtain the desired ionic composition in the final digestion mixture. The simulated
digestion fluids were autoclaved at 121 ◦ C for 15 min along with the calcium chloride
solution and the water. The enzyme solutions were prepared by mixing the enzyme
powder aseptically in sterile water. All the solutions used during the in vitro digestion
process were stored refrigerated and were prewarmed at 37 ◦ C overnight, apart from the
enzyme solutions that were freshly prepared. Furthermore, in order to improve pancreatin
solubility, the solution was freshly prepared and stirred at 400 rpm on ice for 2 h.
2.8. In Vitro Digestion Model System

The INFOGEST static in vitro digestion standardized protocol was used to mimic
the human digestion in the upper GIT after applying some minor adaptations [17]. A
schematic representation of this study’s experimental process is demonstrated in Figure 1.
The simulated digestion process consisted of an oral, a gastric and an intestinal phase, with
the latter corresponding to the proximal small intestinal segments, duodenum and jejunum.
Temperature control was achieved by placing the digestion set-up in an incubator at 37 ◦ C,
and mixing of the digesta was conducted by stirring at 400 rpm. The control of pH was
accomplished with the addition of the appropriate amounts of 3 M HCl (ThermoFisher
Scientific, MA, USA) or 1 M NaOH (VWR, PA, USA), and their required volumes were
pre-determined for each step of digestion in a test prior to digestion experiments. Briefly,
during the oral phase, the developed FMS was mixed with SSF at a ratio of 1:1 (w/w),
including 500 µL of 0.3 M CaCl2 (H2 O)2 , the enzyme solution and sterile water, to achieve a
1× concentration of the SSF. In fact, after the pH was adjusted to 7.0, 10 mL of a fresh 3%
w/v α-amylase solution was added, aiming to achieve an enzymatic activity of 75 U/mL in
the final mixture. The duration of the oral digestion process was 2 min. Afterwards, the
oral bolus was mixed with SGF at a ratio of 1:1 (vol/vol) that included 100 µL of 0.3 M
CaCl2 (H2 O)2 , the enzyme solution, HCl and sterile water, marking the beginning of the
gastric phase. In specific, 10 mL of a 20% w/v porcine pepsin solution was added in order
to achieve a final enzymatic activity of 2000 U/mL in the gastric chyme. After the addition
of pepsin, the pH of the gastric phase was adjusted to 2.5 for the digestion experiments
focusing on S. Typhimurium and to pH 3.0 for L. monocytogenes. The difference in the
gastric acidity when investigating the behavior of the two pathogens can be explained by
the need to apply similar microbial acidic shock and the difference in pH resistance of the
two microorganisms as established in an earlier study [16]. The duration of the gastric
phase was 2 h, and it was followed by the intestinal phase, in the beginning of which
the gastric chyme was mixed with SIF at a ratio of 1:1 (vol/vol). Then, the digesta was
further supplemented with 800 µL of 0.3 M CaCl2 (H2 O)2 , 50 mL of a 16% w/v porcine bile
extract solution (10 g/L in the final mixture), 8 mL of a 10% w/v L-cysteine hydrochloride
monohydrate solution (Alfa Aesar, MA, USA), 800 µL of a 50% w/v resazurin solution (Alfa
Aesar, MA, USA) and sterile water (attaining a 1× concentration of the SIF). The addition
of L-cysteine hydrochloride and resazurin served for the reduction in the redox potential
in the mixture and for monitoring the changes in the redox potential, respectively [18,19].
With respect to the intestinal pH, during the first hour of intestinal digestion, it was adjusted
to 5.5, mimicking as such the duodenal acidic conditions, and during the second hour of
intestinal digestion, it was fixed to 6.6, simulating the jejunal acidic conditions. After the
pH adjustment, 100 mL of a 0.38% w/v pancreatin solution was added to attain a final
trypsin activity of 100 U/mL. Lastly, the digesta was flushed with anoxic nitrogen gas6 for
Microorganisms 2023, 11, x FOR PEER REVIEW of 19

the whole duration of the intestinal phase, given the necessity for anaerobic conditions
during the intestinal phase.

Figure 1. Schematic overview of the implemented experimental protocol.
Figure 1. Schematic overview of the implemented experimental protocol.
2.9. Hurdle Technology Application and Microbiological Analysis

2.9. Hurdle Technology Application and Microbiological Analysis
As an initial step, each FMS with a specific pH–aw combination was inoculated with an
As an initial step, each FMS with a specific pH–aw combination was inoculated with
infectious dose of 109 CFU of either S. Typhimurium or L. monocytogenes, and it was stored
an infectious dose of 10 9 CFU of either S. Typhimurium or L. monocytogenes, and it was
at 10 ◦ C or 25 ◦ C for 6 days, depending on the hurdle combination under investigation.
stored at 10 °C or 25 °C for 6 days, depending on the hurdle combination under investi‐
gation. After the habituation of each pathogen to the appropriate conditions, the in vitro
digestion process was conducted as previously described. Samples were obtained at dis‐
tinct time intervals during the oral, gastric and intestinal phase in order to characterize
microbial growth dynamics. Serial decimal dilutions of the samples were prepared in
After the habituation of each pathogen to the appropriate conditions, the in vitro digestion
process was conducted as previously described. Samples were obtained at distinct time
intervals during the oral, gastric and intestinal phase in order to characterize microbial
growth dynamics. Serial decimal dilutions of the samples were prepared in 0.85% w/v
NaCl solution and then inoculated on solid nutritional media. The inoculation of the plates
was performed either by transferring three drops of 20 µL or by surface plating 100 µL of
the appropriate dilution. For S. Typhimurium, TSA was used as a solid nutritional medium,
whilst for L. monocytogenes, BHI agar was selected. The plates were incubated at 37 ◦ C for
24–48 h and by the enumeration of colonies on the incubated plates, the cell density was
determined. The number of biological replicates was two.
2.10. Modelling Microbial Kinetics

The model shown in Equations (1)–(3) was fitted to the experimental data obtained
from the in vitro digestion process for both the gastric and the intestinal phases. This model
describes a microbial inactivation curve consisting of a shoulder, a log-linear inactivation
phase and a tail [20].

dN(t) 1 Nres
=− ·rmax · 1 − ·N(t) (1)
dt 1 + C(t) N(t)
dC(t)
= −rmax ·C(t) (2)
dt
ln(1 + C0 )
SL = (3)
rmax
where N(t) (CFU) is the cell density at time t (min), rmax (1/min) is the maximum specific
inactivation rate, Nres (CFU) is the residual population in the tail phase, C(t) (−) is a
measure of the physiological state of the cells and SL is the shoulder length (min). The
initial conditions at t = 0 are denoted by N0 (CFU) for the initial cell density and C0 (−) for
the initial physiological state of the cells. By omitting the first and/or the third factor of
Equation (2), the model excluded the shoulder and/or the tailing phase when they were
absent. The lsqnonlin routine of the Optimization Toolbox of MATLAB version R2015b
(The Mathworks, Inc., Natick, MA, USA) was used for the minimization of the sum of
squared errors to estimate the parameters of the mathematical model. Lastly, the parameter
estimates were determined based on the Jacobian matrix [21] and the goodness of the model
fit was based on the Mean Squared Error (MSE). For each dataset, the factors included in
the model (shoulder/tail) were selected to obtain the lowest MSE with the fewest model
parameters. When the estimated rate rmax < 0, it represents the maximum specific growth
rate, and when rmax > 0, it represents the maximum specific inactivation rate.
2.11. Statistical Analysis

The analysis of variance (ANOVA) test was performed to determine whether there are
any significant differences among means of logarithmically transformed viable counts, at
a 95.0% confidence level (α = 0.05). The Tukey’s honestly significant difference (Tukey’s
HSD) test was used for pairwise comparison of the ANOVA results. The analyses were
performed using the anova1 routine of the Statistical Toolbox of MATLAB version R2018b.
Test statistics were regarded as significant when p ≤ 0.05.
3. Results and Discussion

3.1. Assessment and Modelling of Growth Boundaries
Initially, the growth limits of Salmonella Typhimurium and Listeria monocytogenes were
investigated in several different combinations of aw , pH and storage temperature of the
developed FMS, as explained in Section 2.4. The reason for this was to select the appropriate
hurdle combinations, to which each pathogen would be habituated prior to the in vitro
digestion experiments.
The results showed that for S. Typhimurium at 10 ◦ C and 25 ◦ C, the minimum aw

values for growth were 0.982 and 0.949, respectively, and the minimum pH values for
growth were 5.0 and 4.2, respectively (Figure 2a,b). On the other hand, L. monocytogenes ap-
peared more tolerant to the inimical environmental stresses, where the minimum aw value
for growth was 0.931 at both temperatures and the minimum pH values that permitted
growth were 4.6 at 10 ◦ C and 4.2 at 25 ◦ C (Figure 2c,d). Similarly, previous studies have
illustrated that S. Typhimurium required a higher minimum aw value for growth (aw 0.942)
in a liquid broth at 25–35 ◦ C than L. monocytogenes (aw 0.900). Nevertheless, the minimum
pH value for growth in the same conditions was higher for L. monocytogenes with a value
of 4.45 compared with S. Typhimurium that required a minimum pH of 3.94 [22,23]. In
general, comparing the boundaries of growth of the two bacteria, it is quite evident that
L. monocytogenes exhibited broader margins of growth than S. Typhimurium, especially
when the storage temperature was at 10 ◦ C. These observations were quite expected, given
that L. monocytogenes experienced less of an effect on the growth boundaries of aw and
pH due to the decreased temperature, being a psychrophile [24]. Additionally, for both
023, 11, x FOR PEER REVIEW microorganisms, there were a few examples of conditions close to the growth/no growth
8 of 19
boundary where some replicates grew and others did not, being signified as combinations
that had partial growth.

Figure 2. Growth/no growth interface of (a) S. Typhimurium at 10 ◦ C, (b) S. Typhimurium at 25 ◦ C,
Figure 2. Growth/no growth interface of (a) S. Typhimurium at 10 °C, (b) S. Typhimurium at 25 °C,
(c) L. monocytogenes at 10 ◦ C and (d) L. monocytogenes at 25 ◦ C, with respect to pH and
(c) L. monocytogenes at 10 °C and (d) L. monocytogenes at 25 °C, with respect to pH and a w predicted
aw predicted by
by logistic regression model and compared with the data used to generate the model (x stands for
logistic regression model and compared with the data used to generate the model (x stands for growth,
growth, x stands for partial growth, x stands for no growth, the black dashed line for the 50% chance
x stands for partial growth, x stands for no growth, the black dashed line for the 50% chance of growth
and the red circles denote the hurdle combinations selected for the in vitro digestion experiments).
of growth and the red circles denote the hurdle combinations selected for the in vitro digestion ex‐
periments).
Remarkably, the results illustrated that the probability of growth may be altered
dramatically by slight changes in aw and pH, as has been previously demonstrated for
Remarkably, the results illustrated that the probability of growth may be altered dra‐
S. Typhimurium, L. monocytogenes and E. coli [22,23,25]. In a similar manner, storage
matically by slight changes played
temperature in aw aand pH,
crucial roleas
inhas been previously
the growth limits of each demonstrated
pathogen, where,for S.
as expected,
◦
Typhimurium, L. monocytogenes and E. coli [22,23,25]. In a similar manner, storage temper‐
a higher temperature (25 C) enabled more combinations of aw and pH to permit growth.
The latter indicates a synergistic effect of aw , pH and temperature on the growth limits of
ature played a crucial role in the growth limits of each pathogen, where, as expected, a
the two pathogens that is, in fact, observed in all of the investigated conditions. However,
higher temperature (25 °C) enabled more combinations of a w and pH to permit growth.
a previous study on the boundaries of growth of S. Typhimurium in tryptic soy broth
The latter indicates a synergistic effect of aw, pH and temperature on the growth limits of
the two pathogens that is, in fact, observed in all of the investigated conditions. However,
a previous study on the boundaries of growth of S. Typhimurium in tryptic soy broth
exhibited a non‐synergistic effect of the same hurdles when the aw ranged between 0.990
and 0.955 [22]. A putative explanation for the inconsistency of these findings with our
exhibited a non-synergistic effect of the same hurdles when the aw ranged between 0.990 and
0.955 [22]. A putative explanation for the inconsistency of these findings with our results
can be that the current examined system, i.e., the FMS, was a viscous food model system
rather than a liquid broth. In fact, several previous studies have illustrated differences
between the growth limits of bacteria in solid and liquid media, indicating the significance
of the medium’s state [23,26,27].
From the overall observation of the results, a total of eight different combinations of
temperature (10 ◦ C, 25 ◦ C), pH (5.0, 6.6) and aw (0.909 or 0.931, 0.994) were selected for the
preparation and storage of the developed FMS. These combinations are clearly denoted in
Figure 2 with the red circles. Out of these conditions, half belong to the growth region and
the other half belong to the no-growth region, for both pathogens. The conditions represent-
ing the no-growth regions of each pathogen were chosen for their ability to inhibit growth
but not to significantly inactivate the microbial population. S. Typhimurium expressed
different and more narrow boundaries of growth than L. monocytogenes. Consequently, the
selected combinations of hurdles for each bacterium differed, primarily in the lower value
of aw , which was 0.931 for S. Typhimurium and 0.909 for L. monocytogenes.
3.2. Effect of the Hurdle Technology Application on the Survival of Salmonella Typhimurium and
Listeria Monocytogenes
3.2.1. Salmonella Typhimurium
1. General remarks
The microbial kinetics of S. Typhimurium are observed in Figure 3A–H, during each
phase of simulated digestion. Table 1 illustrates the parameter estimates of the microbial
kinetics of the pathogen obtained from the inactivation model [20], along with the Mean
Squared Error (MSE) and the final log reduction. A general observation of the results
revealed a linear trend during the intestinal phase for all eight experimental conditions
selected and a more complex behavior during the gastric phase. More specifically, the
results demonstrated inactivation of the pathogen during the gastric phase and less in-
activation or even growth during the intestinal phase in all experimental conditions [16].
This phenomenon can be mainly attributed to the elevated sensitivity of S. Typhimurium
toward the low values of gastric pH (2.5), along with its high tolerance against intestinal bile
acids [10,28]. The great antimicrobial effects that gastric pH can infer on S. Typhimurium
have been also previously demonstrated, where S. Typhimurium exhibited higher acid
sensitivity than L. monocytogenes and E. coli when their microbial kinetics were compared
under various pH values of simulated gastric fluid [28]. In addition, this finding is further
corroborated by two of our previous studies, in which S. Typhimurium expressed similar
behavior during the simulated digestion process when investigating the effects of gastric
pH and intestinal bile acids or the effect of the food carrier properties on its survival during
simulated digestion [16,29].
With respect to the different hurdle combinations, they were distinguished in growth
and no growth environmental conditions, with the hurdle combinations with an aw of 0.994
permitting the growth of S. Typhimurium in the FMS (growth region) and those with an
aw of 0.931 inhibiting the growth of the pathogen (no growth region) (Figure 3). In fact,
previous research has revealed that the minimum aw for the growth of S. Typhimurium is
0.94, further corroborating our findings [30]. Hence, as illustrated from the results, even
though the inoculum level in all conditions was 109 CFU, in the beginning of digestion
(t = 0 min), the initial population of S. Typhimurium had increased at conditions A–D
and reduced at conditions E–H. For instance, in condition C, the initial population of
S. Typhimurium was 11.347 log(CFU) (Figure 3C), while in condition F, the initial bacterial
cell density was significantly lower with a value of 5.887 log(CFU) (Figure 3F).
2. Hurdles during in vitro digestion
The results revealed that the hurdle combination with the lowest inactivation was
condition D (aw 0.994–pH 5.0–10 ◦ C), with the final log reduction (throughout the whole
1. General remarks
The microbial kinetics of S. Typhimurium are observed in Figure 3A–H, during each
phase of simulated digestion. Table 1 illustrates the parameter estimates of the microbial
Squared Error (MSE) and the final log reduction. A general observation of the results re‐
vealed a linear trend during the intestinal phase for all eight experimental conditions se‐
lected and a more complex behavior during the gastric phase. More specifically, the re‐
sults demonstrated inactivation of the pathogen during the gastric phase and less inacti‐
duration of simulated digestion) being the lowest (1.303 log(CFU)) and with a residual
vation or even growth during the intestinal phase in all experimental conditions [16]. This
population at the end of the gastric phase (6.202 log(CFU)) (Figure 3D). In contrast, the
phenomenon can be mainly attributed to the elevated sensitivity of S. Typhimurium to‐
hurdle combination that was related to the highest inactivation of S. Typhimurium during
ward the low values of gastric pH (2.5), along with its high tolerance against intestinal bile
digestion was condition F (aw 0.931–pH 5.0–25 ◦ C), which inferred the highest final log
acids [10,28]. The great antimicrobial effects that gastric pH can infer on S. Typhimurium
reduction in the pathogen with a value of 6.629 log(CFU) (Figure 3F). As such, condition D
have been also previously demonstrated, where S. Typhimurium exhibited higher acid
was associated with increased bacterial stress tolerance, which is a phenomenon that could
sensitivity than L. monocytogenes and E. coli when their microbial kinetics were compared
be attributed to the “stress-hardening” phenomenon triggered by the previous exposure
under various pH values of simulated gastric fluid [28]. In addition, this finding is further
of pathogen to a low aw and low pH in the FMS that eventually enabled its survival in
corroborated by two of our previous studies, in which S. Typhimurium expressed similar
the acidic environment of the simulated stomach [7]. To obtain a better understanding
behavior during the simulated digestion process when investigating the effects of gastric
of how the different hurdle combinations in the FMS affected the survival patterns of
pH and intestinal bile acids or the effect of the food carrier properties on its survival dur‐
S. Typhimurium during digestion, an elaborate discussion is included, focusing each time
ing simulated digestion [16,29].
on a single hurdle’s impact.

Figure 3. Effect of hurdle combinations of water activity (a ), pH and storage temperature on the
Figure 3. Effect of hurdle combinations of water activity (aw w), pH and storage temperature on the
microbial kinetics of S. Typhimurium during simulated digestion. The different phases of in vitro
microbial kinetics of S. Typhimurium during simulated digestion. The different phases of in vitro
digestion, i.e., the oral phase, the gastric phase and the intestinal phase, are differentiated by a line and
digestion, i.e., the oral phase, the gastric phase and the intestinal phase, are differentiated by a line
by the colors orange (left), light green (middle) and light blue (right), respectively. The experimental
and by the colors orange (left), light green (middle) and light blue (right), respectively. The experi‐
data are signified by the circles, while the lines represent the fit of the model for inactivation [20] to
mental data are signified by the circles, while the lines represent the fit of the model for inactivation
[20] to the data. The colors green and purple distinguish the two different replicates.
the data. The colors green and purple distinguish the two different replicates.

Hurdle of aw
At the conditions where the pH and the storage temperature of the FMSs were similar,
but the aw differed, a significant finding that was observed was that a low aw in the
FMS (0.931) was often associated with increased inactivation of S. Typhimurium during
simulated gastric digestion, while an aw of 0.994 inferred the tolerance of the pathogen
against gastric acidity. For instance, observing the final log reduction values of conditions
C (Figure 3C, aw 0.994–pH 5.0–25 ◦ C) and G (Figure 3G, aw 0.931–pH 5.0–25 ◦ C), it is quite
clear that microbial inactivation was significantly higher at condition G (gastric rmax +0.031
1/min and final log reduction 6.629 log(CFU)) than at condition C (gastric rmax +0.009
1/min and final log reduction 2.067 log(CFU)). As such, our results revealed that in the case
of acidic stress (gastric acidity), a previous exposure to a low aw was able to induce the acid
tolerance of S. Typhimurium. Previous research concerning the acid responses of Salmonella
after exposure to low aw foods is limited, yet the prolonged survival of Salmonella in low aw
values has been observed to induce the tolerance of the pathogen against heat [31–33].
Table 1. Kinetic parameters estimates of the inactivation model [20], both for the simulated gastric phase and the simulated intestinal phase, obtained for the cells of
S. Typhimurium during the in vitro digestion of the different food model systems 1,2 .
Gastric Phase Intestinal Phase

Final Log
Experimental Condition rmax (1/min) Nres [log(CFU)] MSE rmax (1/min) Reduction
[log(CFU) ± SD]
95% 95%
Confidence Confidence
Interval Interval
A aw 0.994–pH 6.6–25 ◦ C +0.012 a [+0.009, +0.015] - - 0.107 +0.003 a [+0.001, +0.006] 2.023 ± 0.167 a
B aw 0.994–pH 6.6–10 ◦ C +0.052 b [+0.041, +0.062] 6.931 a [6.716, 7.147] 0.128 −0.004 b [−0.003, −0.001] 2.497 ± 0.199 b
C aw 0.994–pH 5.0–25 ◦ C +0.009 c [+0.007, +0.011] - - 0.070 +0.007 c [+0.005, +0.009] 2.067 ± 0.132 a
D aw 0.994–pH 5.0–10 ◦ C +0.095 d [+0.072, +0.019] 6.202 a [5.933, 6.467] 0.285 −0.012 d [−0.018, −0.007] 1.303 ± 0.265 c
E aw 0.931–pH 6.6–25 ◦ C +0.026 g [+0.015, +0.037] 3.288 b [0.236, 6.341] 0.495 −0.004 b [−0.012, +0.004] 2.251 ± 0.287 b
F aw 0.931–pH 6.6–10 ◦ C +0.012 a [+0.010, +0.013] - - 0.045 +0.004 e [+0.002, +0.005] 1.962 ± 0.103 a
G aw 0.931–pH 5.0–25 ◦ C +0.031 f [+0.024, +0.038] - - 0.754 +0.021 f [+0.014, +0.028] 6.629 ± 0.423 d
H aw 0.931–pH 5.0–10 ◦ C +0.015 e [+0.006, +0.023] - - 0.988 +0.004 e [+0.003, +0.011] 2.408 ± 0.492 b
1The superscript lowercase letters indicate the differences obtained by ANOVA between the same kinetic parameters for each experimental condition. Parameter estimates that do not
share the same letter are significantly different (p < 0.05). 2 The rmax estimates indicate growth when rmax < 0, or inactivation when rmax > 0.
Another important finding was that the effect of aw on the acid stress responses of
S. Typhimurium during gastric digestion was greatly dependent on the FMS’s storage tem-
perature, indicating a synergistic or additive effect. More specifically, when the aw was 0.994,
a storage temperature of 10 ◦ C resulted in the extensive inactivation of S. Typhimurium
during the gastric phase (Figure 3B,D), where the highest gastric rmax values were ob-
served, i.e., +0.052 1/min for condition B (aw 0.994–pH 6.6–10 ◦ C) and +0.095 1/min for
condition D (aw 0.994–pH 5.0–10 ◦ C). On the contrary, when the aw of the FMS was low
and at the no-growth region of S. Typhimurium (0.931), a storage temperature of 10 ◦ C
was associated with a more acid-tolerant microbial behavior during simulated gastric
digestion (Figure 3F,H). For example, the values of gastric rmax for conditions F and H were
significantly lower (+0.015 and +0.012 1/min, respectively) when compared to conditions
E and G (+0.031 and 0.026 1/min, respectively), in which the storage temperature was
higher (25 ◦ C). Salmonella is a mesophilic bacterium with an optimal temperature range of
30–45 ◦ C and with the minimum temperature for growth being 5.2 ◦ C [30,34]. Thereby, the
habituation of S. Typhimurium to hurdle combinations at the growth region (aw 0.994) with
a storage temperature as low as 10 ◦ C presumably inflicted cell damage to the pathogen
that caused, in turn, its significant inactivation during the acidic gastric phase. In contrast,
when S. Typhimurium was previously exposed to the same temperature at 10 ◦ C but at the
no-growth region (aw 0.931), the simultaneous application of multiple sublethal stresses
triggered its “stress hardening”, resulting in the pathogen’s elevated acid tolerance against
gastric acidity.
Hurdle of pH
At the conditions where the aw and the storage temperature of the FMSs were the
same but the pH was different, the results showed a diverged microbial behavior of
S. Typhimurium between the conditions that were at the growth region and those that were
at the no-growth region, indicating the influence of aw exposure on the antimicrobial effects
of gastric pH.
When the aw was at the growth region of the pathogen (0.994), a low pH value in the
FMS resulted in the increased tolerance of S. Typhimurium against gastric acidity when
compared to a higher pH value. For example, the highest inactivation of the pathogen was
observed at condition B (aw 0.994–pH 6.6–10 ◦ C) when the pH was high (pH 6.6) (Figure 3B)
with a final log reduction of 2.497 log(CFU). Yet, when the pH of the FMS was lower
(Figure 3D, aw 0.994–pH 5.0–10 ◦ C), S. Typhimurium demonstrated a significantly tolerant
behavior against gastric acidity with a final log reduction of 1.303 log(CFU), which was, in
fact, the lowest log reduction reported in this study’s results. In fact, acid habituation of
S. Typhimurium has been previously associated with increased microbial tolerance toward
several sources of otherwise lethal stress, such as heat, acidity and osmotic stress, with
the literature suggesting that a pH range of 4.0–5.0 may infer excessive resistance to this
bacterium [9,35,36].
On the other hand, when the aw was at the no-growth region of S. Typhimurium (0.931),
a low pH value in the FMS (pH 5.0) caused the extensive inactivation of the pathogen during
simulated digestion, while a higher pH value (6.6) was associated with a more tolerant
microbial behavior. For instance, in condition G (Figure 3G, aw 0.931–pH 5.0–25 ◦ C), a pH
of 5.0 contributed to the highest final reduction in the pathogen, along with a significantly
higher gastric rmax (+0.031 1/min), when compared to the rmax value of +0.026 1/min
obtained for condition E (Figure 3E, aw 0.931–pH 6.6–25 ◦ C). The association of the higher
pH values with increased acid tolerance is also illustrated by the limited final log reduction
values, e.g., 2.251 log(CFU) for condition E and 1.962 log(CFU) for condition F, as well as by
the presence of a residual population that was often observed. The exposure of Salmonella
to mild acidity may activate the ATR of the pathogen that can, in turn, enable its survival
at otherwise lethal conditions [8]. The latter, in combination with the growth-inhibiting
levels of aw in the FMS, could, in fact, induce the ATR of S. Typhimurium. The combined
effect of low aw with other sublethal stresses on microbial survival has also been previously
demonstrated when Salmonella Typhimurium expressed a greater acid tolerance in SGF after
its previous exposure to a low aw and mildly acidic dry-cured meat product at 25 ◦ C [9].
Hurdle of Storage Temperature

When focusing on the effect of storage temperature, the results showed that when the
FMS was stored at a higher temperature (25 ◦ C), S. Typhimurium was greatly inactivated
during simulated digestion, whilst a storage temperature of 10 ◦ C was associated with
limited inactivation of the pathogen. This finding was rather expected, since bacteria
have the capacity to synthesize cold-shock proteins to hinder the lethal effects of cold
shock [37]. For instance, S. Typhimurium has been reported to produce the cold-shock
protein B (CspB), as a response to temperature decreases below 24 ◦ C [38]. In addition, it
has been revealed that CspB can widely promote oxidative, pH, osmotic, starvation and
ethanol stress tolerance, playing, as such, an active role in bacterial cross-protection [39].
Hence, when comparing the microbial kinetics of S. Typhimurium after habituation to the
growth-inhibiting conditions G and H, the final log reduction and the gastric rmax were
higher in condition G (Figure 3G, aw 0.931–pH 5.0–25 ◦ C), where the storage temperature
was 25 ◦ C (6.629 log(CFU), +0.031 1/min, respectively), than in condition H (Figure 3H,
aw 0.931–pH 5.0–10 ◦ C), where the storage temperature was 10 ◦ C (2.408 log(CFU), +0.015
1/min, respectively). In fact, condition H corresponds to the harshest environmental
conditions for microbial survival; nonetheless, S. Typhimurium exhibited a relatively
limited final log reduction in its population, as well as the second lowest gastric rmax value,
indicating an adaptive tolerance of the pathogen to the gastricidal effects of the simulated
stomach. Interestingly, the different levels of aw and pH did not exhibit a considerable
impact on the general effect of storage temperature on the behavior of S. Typhimurium
during simulated digestion. Thereby, when at the growth region, a habituation of the
pathogen to conditions with a low storage temperature resulted as well in increased
microbial tolerance, with an apparent residual population and proliferation during the
simulated intestinal phase. For instance, the habituation of S. Typhimurium to conditions B
(Figure 3B, aw 0.994–pH 6.6–10 ◦ C) and D (Figure 3D, aw 0.994–pH 5.0–10 ◦ C) manifested
a similar residual population of 6.931 log(CFU) and 6.202 log(CFU), respectively, as well
as proliferation during intestinal digestion with an rmax of −0.004 1/min and of −0.012
1/min, respectively.
3.2.2. Listeria monocytogenes

1. General remarks
Figure 4A–H demonstrate the microbial kinetics of L. monocytogenes during each
phase of the in vitro digestion. Table 2 shows the parameter estimates of the microbial
Squared Error (MSE) and the final log reduction. A general trend that was observed
was a more complex behavior during the intestinal phase, whereas the gastric phase was
mainly dictated by linearity. In addition, the results revealed a high acid tolerance of
L. monocytogenes during the acidic gastric phase (pH 3.0) and sensitivity of the pathogen
to the bactericidal effects of intestinal bile acids [16]. A previous study has illustrated
that L. monocytogenes was characterized by a greater acid tolerance than S. Typhimurium
when they were both subjected to an acid challenge test with lactic acid (pH 3.5) [36]. In
addition, during the simulated intestinal phase, L. monocytogenes was mainly inactivated
due to the bactericidal effects of bile acids, and a residual population was often observed.
These findings are in concordance with previous studies, where L. monocytogenes expressed
a similar behavior when the effect of gastric pH and intestinal bile acids or the effect of
food-buffering capacity and food composition on its survival were under investigation
during in vitro digestion [16,29]. In fact, the apparent sensitivity of L. monocytogenes to
bile acids could be explained by the pathogen’s previous exposure to gastric acidity that
can infer a subsequent susceptibility to the bactericidal properties of bile acids [16]. In
addition, previous findings have exhibited that bile sensitivity could be pH-dependent
Microorganisms 2023, 11, x FOR PEER REVIEW 14 of 19

when L. monocytogenes strains manifested a decreased survival after their exposure to bile
acids at a pH value of 5.5, which is a case similar to this study [40].

Figure 4. Effect of hurdle combinations of water activity (aw ), pH and storage temperature on the
Figure 4. Effect of hurdle combinations of water activity (aw), pH and storage temperature on the
microbial kinetics of L. monocytogenes during simulated digestion. The different phases of in vitro
microbial kinetics of L. monocytogenes during simulated digestion. The different phases of in vitro
digestion, i.e., the oral phase, the gastric phase and the intestinal phase, are differentiated by a line and
digestion, i.e., the oral phase, the gastric phase and the intestinal phase, are differentiated by a line
by the colors orange (left), light green (middle) and light blue (right), respectively. The experimental
and by the colors orange (left), light green (middle) and light blue (right), respectively. The experi‐
mental data are signified by the circles, while the lines represent the fit of the inactivation model
data are signified by the circles, while the lines represent the fit of the inactivation model [20] to the
[20] to the data. The colors green and purple distinguish the two different replicates.
data. The colors green and purple distinguish the two different replicates.
Table 2. Kinetic parameters estimates of the inactivation model [20], both for the simulated gastric
Concerning the different experimental conditions, they were distinguished in growth
phase and the simulated intestinal phase, obtained for the cells of Listeria monocytogenes during the
and no growth conditions, as previously explained. Consequently, conditions A–D were in
in vitro digestion of the different food model systems
the growth region, where, as illustrated in Figure 4A–D,
1,2.
the initial microbial cell densities of
L. monocytogenes in the beginning of digestion and after its habituation to the different envi-
Gastric Phase Intestinal Phase Final Log
ronmental conditions were higher than 109 CFU, which was the FMS’s inoculum level (e.g.,
Experimental Reduction
at condition A the N0 was 10.808 log(CFU)). On the other hand, conditions E–H characterize
Condition rmax (1/min) [log(CFU) ±
the hurdle combinationsrmax (1/min)
that did not permit theNgrowth
res [log(CFU)] MSE (Figure
of L. monocytogenes 4E–H);
SD]
hence, the initial population levels of the pathogen did not increase significantly. For
instance, as observed at condition E, the N0 was 9.172 95% Confi‐
95% Confi‐ log(CFU).
95% Confi‐
2. Hurdles during in vitro dence Inter‐
dence Interval digestion dence Inter‐
val val
The hurdle combination that led to the highest subsequent inactivation of L. monocyto-
aw 0.994– genes during simulated digestion was condition E (aw 0.909–pH ◦
+0.003 [+0.013, [3.388, 6.6–25 C), where2.728 ±
the final
A pH 6.6– [+0.002, +0.003] +0.018 a
log reduction in the pathogen was 6.698 a
estimated at 4.032 log(CFU) 0.066
a +0.024] 10.009] and the intestinal 0.146
rmax awas

25 °C +0.055 1/min, rendering the highest estimated values among the different experimental
aw 0.994– conditions (Figure 4E). On the other side, the lowest inactivation was obtained at hurdle
+0.012 combination C (aw 0.994–pH [+0.005,
5.0–25 [4.550,
◦ C), where the final log reduction 0.120 3.303 ±
in the pathogen was
B pH 6.6– b
[+0.009, +0.015] +0.016 b 7.000 a
+0.027] 9.450]
significantly lower than the rest of the cases, with a value of 1.657 log(CFU), indicating 0.225 b an
10 °C
acquired microbial tolerance of L. monocytogenes to the bactericidal effects of intestinal bile
aw 0.994– acids (Figure 4C). Indeed, L. monocytogenes has exhibited the capacity to express1.657 ±
adaptive
+0.003 [+0.005,
C pH 5.0– a
[+0.002, +0.004] +0.010 c
stress responses via the process +0.001] ‐ 0.167
of “stress hardening” after being exposed to sublethal levels
0.177 c
25 °C of acidity (pH 5.0–5.5), that can, in turn, enhance its capacity to survive its passage through
aw 0.994– the human GIT [41,42]. The interpretation of the results is organized in the same manner as
+0.006 for S. Typhimurium, focusing [+0.009,
each time on the effects ‐ of one individual hurdle. 1.939 ±
D pH 5.0– c
[+0.006, +0.007] +0.010 c 0.030
+0.011] 0.077 d
10 °C
aw 0.909–
+0.004 [+0.045, 4.032 ±
E pH 6.6– d
[+0.001, +0.007] +0.055 g 5.115 b [4.783, 5.448] 0.150
+0.065] 0.189 g
25 °C
Table 2. Kinetic parameters estimates of the inactivation model [20], both for the simulated gastric phase and the simulated intestinal phase, obtained for the cells of
Listeria monocytogenes during the in vitro digestion of the different food model systems 1,2 .
Gastric Phase Intestinal Phase Final Log Reduction

Experimental Condition
rmax (1/min) rmax (1/min) Nres [log(CFU)] MSE [log(CFU) ± SD]
95% 95% 95%
Confidence Confidence Confidence
Interval Interval Interval
A aw 0.994–pH 6.6–25 ◦ C +0.003 a [+0.002, +0.003] +0.018 a [+0.013, +0.024] 6.698 a [3.388, 10.009] 0.066 2.728 ± 0.146 a
B aw 0.994–pH 6.6–10 ◦ C +0.012 b [+0.009, +0.015] +0.016 b [+0.005, +0.027] 7.000 a [4.550, 9.450] 0.120 3.303 ± 0.225 b
C aw 0.994–pH 5.0–25 ◦ C +0.003 a [+0.002, +0.004] +0.010 c [+0.005, +0.001] - 0.167 1.657 ± 0.177 c
D aw 0.994–pH 5.0–10 ◦ C +0.006 c [+0.006, +0.007] +0.010 c [+0.009, +0.011] - 0.030 1.939 ± 0.077 d
E aw 0.909–pH 6.6–25 ◦ C +0.004 d [+0.001, +0.007] +0.055 g [+0.045, +0.065] 5.115 b [4.783, 5.448] 0.150 4.032 ± 0.189 g
F aw 0.909–pH 6.6–10 ◦ C +0.002 e [+0.001,+0.004] +0.041 f [+0.028, +0.053] 6.584 a [6.238, 6.931] 0.123 2.573 ± 0.158 f
G aw 0.909–pH 5.0–25 ◦ C +0.004 d [+0.002,+0.006] +0.033 e [+0.017, +0.049] 6.374 a [6.459, 7.009] 0.070 2.216 ± 0.131 e
H aw 0.909–pH 5.0–10 ◦ C +0.003 a [+0.002,+0.004] +0.013 d [+0.012, +0.015] - 0.030 2.147 ± 0.075 e
1The superscript lowercase letters indicate the differences obtained by ANOVA between the same kinetic parameters for each experimental condition. Parameter estimates that do not
share the same letter are significantly different (p < 0.05). 2 The rmax estimates indicate growth when rmax < 0, or inactivation when rmax > 0.
Hurdle of aw
When the hurdles of pH and storage temperature were alike, but the aw levels differed,
the results illustrated that in most cases, a lower aw was responsible for the subsequent
elevated inactivation of L. monocytogenes during intestinal digestion, while a higher aw was
often associated with microbial tolerance. For instance, after the pathogen was habituated
to condition A (Figure 4A, aw 0.994–pH 6.6–25 ◦ C), its population exhibited a significantly
lower final log reduction (2.728 log(CFU)) and a significantly lower intestinal rmax (+0.018
1/min) during simulated digestion when compared to condition E (aw 0.909–pH 6.6–25 ◦ C)
(Figure 4E), that, as previously explained, exhibited the highest values of final log reduction
and intestinal rmax . Overcoming osmotic stress is considered an energy-depleting process
for bacteria, since great amounts of metabolic energy are required to accumulate compatible
solutes intracellularly in order to limit water loss [43,44]. Thereby, the continuous energy
depletion due to the exposure to a series of subsequent stresses, e.g., osmotic stress, acid
stress in the stomach, and bile acids toxicity in the intestine, could explain the increased
inactivation of L. monocytogenes after its habituation to sublethal levels of aw . Interestingly,
the lowest inactivation of L. monocytogenes that was observed at condition C (aw 0.994–pH
5.0–25 ◦ C) was at the growth region of the pathogen (Figure 4C), and it could indicate a
plausible microbial tolerance to the antimicrobial effects of digestion.
Hurdle of pH
Turning now to the impact of pH on microbial behavior, the results demonstrated
that when the hurdles of aw and storage temperature were similar, a lower pH value was
associated with a higher microbial tolerance compared with a higher pH. More specifically,
when the hurdle combinations permitted the growth of L. monocytogenes (aw 0.994), the final
log reduction in the pathogen was higher when the pH was 6.6. Nonetheless, when the pH
was 6.6, a residual population was apparent in all experimental conditions. For instance, in
condition A (Figure 4A, aw 0.994–pH 6.6–25 ◦ C), the final log reduction in L. monocytogenes
at the end of simulated digestion was 2.728 log(CFU), while at condition C (Figure 4C, aw
0.994–pH 5.0–25 ◦ C), it was 1.657 log(CFU), which was the lowest final log reduction among
all cases. Likewise, when comparing conditions F (Figure 4F, aw 0.909–pH 6.6–10 ◦ C) and
H (Figure 4H, aw 0.909–pH 5.0–10 ◦ C), it is quite evident that the final log reduction is
significantly higher in condition F (2.573 log(CFU)) than in condition H (2.147 log(CFU),
along with the intestinal rmax (+0.041 1/min in condition F and +0.013 1/min in condition
H), yet a tail is observed with a value of 6.584 log(CFU) (condition F). These findings can
be attributed to the occurrence of cross-protection, where the low pH of the FMS stressed
the cells of L. monocytogenes, leading to its subsequent hardening on the exposure to the
sublethal stresses of gastrointestinal digestion. In fact, the so-called “stress-hardening”
phenomenon can be associated with the induction of ATR after the previous exposure of the
pathogen to acidic conditions [7,45]. The effects of cross-protection and “stress hardening”
on L. monocytogenes have been previously illustrated, where the pathogen exhibited an
increased acid tolerance to gastric acidity (pH 2.0) after it had been previously habituated
to a pH of 5.5 and 6.6 for 15 days [46]. Moreover, a recent previous study revealed that the
habituation of L. monocytogenes to a pH of 5.5 or 6.0 induced a greater ATR that a pH of
6.5 [47], readily confirming the current findings.
Hurdle of Storage Temperature

When focusing on the storage temperature, a high value (25 ◦ C) during the microbial
habituation to the FMS was related with the increased tolerance of L. monocytogenes against
the bactericidal effects of simulated digestion, whereas a low storage temperature (10 ◦ C)
inferred the pathogen’s increased sensitivity and, as a result, its greater inactivation during
digestion. For instance, the final log reduction in L. monocytogenes after habituation to
condition A (Figure 4A, aw 0.994–pH 6.6–25 ◦ C) was 2.728 log(CFU), whilst in condition B
(Figure 4B, aw 0.994–pH 6.6–10 ◦ C), where the storage temperature was at 10 ◦ C, the final
log reduction was significantly higher and had a value of 3.303 log(CFU). In addition, at
condition A, a significantly lower gastric rmax was observed (+0.003 1/min) in comparison
with the gastric rmax value in condition B (+0.012 1/min). Furthermore, comparing con-
ditions G (Figure 4G, aw 0.909–pH 5.0–25 ◦ C) and H (Figure 4H, aw 0.909–pH 5.0–10 ◦ C),
even though the final log reduction values are similar, it is quite evident that when the
storage temperature of the FMS was at 25 ◦ C, L. monocytogenes exhibited a residual popula-
tion during intestinal digestion (6.374 log(CFU)), indicating obtained microbial tolerance
to the antimicrobial effects of the intestinal bile acids. An explanation for the apparent
lack of stress tolerance of L. monocytogenes after being habituated to conditions where the
environmental temperature was 10 ◦ C could be that given the slower growth rate of the
pathogen at this low temperature, a lesser fraction of its population would be at the late
stationary phase at the end of storage time. Indeed, the exposure of L. monocytogenes to
low temperatures first leads to its cell arrest (acclimation) and then to its adaptation, where
the microbial population is able to proliferate but at a slower rate [48]. Consequently, this
could readily characterize this microbial population as considerably more sensitive to the
subsequent gastrointestinal stresses, since previous studies have clearly demonstrated that
the stationary phase cells are more stress tolerant than the exponential phase ones [49].
4. Conclusions
Hurdle technology is a vastly applied method for food preservation, chiefly depending
on the combined effects of multiple sublethal stresses that eliminate potential microbial
threats in food products or keep them under control. The exposure of microorganisms
to these stresses can sensitize them or harden them toward other types of subsequent
stressful environments, such as the human GIT. The survival of pathogens in the GIT may
be detrimental for a host’s health, as it can lead to infection and disease. The present results
revealed that both S. Typhimurium and L. monocytogenes survived their transit through
the simulated GIT with an increased microbial tolerance often being observed for both
pathogens after their habituation to certain hurdle combinations.
Taking a closer look at the influence of each individual hurdle applied (Figure 5), this
research showed that the habituation of the pathogens to environmental conditions that
did not permit their growth (low aw ) sensitized them toward the antimicrobial effects of
gastrointestinal digestion. In addition, for S. Typhimurium: (i) the hurdle of a low pH was
linked with increased sensitivity to gastric acidity when the aw was low (higher microbial
inactivation) but with increased tolerance when the aw was high (lower microbial inactiva-
tion) and (ii) a low storage temperature caused the increased inactivation of the pathogen,
which was due to adaptive stress responses. On the other hand, for L. monocytogenes, (i) its
habituation to low pH values was related to its decreased inactivation and hence to a
more tolerant behavior and (ii) a low storage temperature of the FMS led to its increased
sensitivity against intestinal bile acids, as indicated by the increased microbial inactivation.
Lastly, in some cases, a synergistic/additive effect was exhibited between certain hurdles,
e.g., aw and pH for S. Typhimurium. It is clear that the selection of hurdle combinations for
optimal food preservation is a complex matter that might lead to unforeseen outcomes for
microbial survival. In fact, stress-tolerant cells may have an increased chance to overcome
the gastrointestinal barriers of the human digestive system, posing a major threat for public
health. Therefore, further research is required to convey a better view of the cross-protection
effects that hurdle technology may infer on bacterial pathogens during their gastrointesti-
nal transit. The future work should draw more attention to (i) the assessment of different
types of hurdles and/or different hurdle combinations that are also commonly used in
the food industry, shedding more light on their potential synergistic effects, and to (ii) the
implementation of an in vitro digestion system that takes into account the complexity of
the real GIT with its antimicrobial barriers, such as the resident gut microbiota.
ing their gastrointestinal transit. The future work should draw more attention to (i) the
assessment of different types of hurdles and/or different hurdle combinations that are also
commonly used in the food industry, shedding more light on their potential synergistic
effects, and to (ii) the implementation of an in vitro digestion system that takes into account
Microorganisms 2023, 11, 405 the complexity of the real GIT with its antimicrobial barriers, such as the resident gut mi‐ 17 of 19
crobiota.

Figure
Figure 5. Overview
5. Overview of microbial
of microbial responses during
responses during in vitro
in vitro digestion,
digestion, focusing
focusing on impact
on the the impact
of a of a
single hurdle.
single hurdle.
Author Contributions: Conceptualization, T.A., S.A., C.S. and J.F.M.V.I.; methodology, T.A., S.A.
and C.S.; software, T.A. and S.A.; validation, T.A. and F.d.M.; formal analysis, T.A.; investigation,
T.A. and F.d.M.; resources, J.F.M.V.I.; data curation, T.A.; writing—original draft preparation, T.A.;
writing—review and editing, S.A, C.S. and J.F.M.V.I.; visualization, T.A.; supervision, J.F.M.V.I.;
project administration, J.F.M.V.I.; funding acquisition, S.A. and J.F.M.V.I. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the KU Leuven Research Fund through project C24/18/046.
Simen Akkermans was supported by the Research Foundation—Flanders under grant 1224620N
and 1224623N.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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microorganisms

Microorganisms 2023, 11, 405. https://doi.org/10.3390/microorganisms11020405 https://www.mdpi.com/journal/microorganisms

2. Materials and Methods

2.2. Inoculum Preparation

2.3. Food Model System Development and Preparation

2.4. Assessment and Modelling of Boundaries of Growth

2.5. Hurdle Technology Application on the Developed FMS

2.6. Enzymes and Bile Acids

2.7. Simulated Digestion Fluids

2.8. In Vitro Digestion Model System

2.9. Hurdle Technology Application and Microbiological Analysis

2.10. Modelling Microbial Kinetics

2.11. Statistical Analysis

3. Results and Discussion

The results showed that for S. Typhimurium at 10 ◦ C and 25 ◦ C, the minimum aw

Gastric Phase Intestinal Phase

Hurdle of Storage Temperature

3.2.2. Listeria monocytogenes

Microorganisms 2023, 11, x FOR PEER REVIEW 14 of 19

Gastric Phase Intestinal Phase Final Log Reduction

Hurdle of Storage Temperature

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